Directed evolution of synthetic riboswitches and a leucyl tRNA synthetaseShow full item record
|Directed evolution of synthetic riboswitches and a leucyl tRNA synthetase
|Doctor of Philosophy
|An orthogonal pair composed by a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum (MtLRS) and tRNA from Halobacterium sp. NRC-1 (Hhl4) was used to incorporate UAAs in bacteria. Using a sequence homology analysis between LeuRS from bacteria and archaea, we determined the amino acid residues responsible for binding in the active site of the MtLRS. We generated three variants containing five mutations in the active site to incorporate 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (Dansyl-Dap), and used these variants in the expression of Z-domain containing an amber stop codon (K7TAG). The variants did not incorporate Dansyl-Dap at the TAG codon of the Z-domain gene. We designed a library of MtLRS variants randomizing the same positions in the active site. For dual genetic selection we constructed the pRCG plasmid, which contains a cat-upp fusion gene with TAG codons at permissible sites of the chloramphenicol acetyl transferase (CAT) gene. Three variations of the pRCG plasmid were tested for amber suppression using the wild type MtLRS lacking the editing domain: Q98TAG, D111TAG, and a double mutant containing both mutations. The Q98TAG showed the highest amber stop codon suppression efficiency for the selection experiment, so this variant was used in the genetic selection of the MtLRS library for the incorporation 4-nitro-1-phenylalanine and Dansyl-Dap. The obtained variants are currently under study to evaluate their ability to incorporate these UAAs to the Z-domain protein. Dual genetic selection was also used to select synthetic riboswitches. As a proof of concept, we randomized eleven bases at the binding site of the synthetic theophylline riboswitch and used the cat-upp fusion gene to select a riboswitch specific for caffeine. The selected variant (CaffRS) was tested for ß-galactosidase activity, a colorimetric assay to study the concentration-signal dependency. We then used this approach to select riboswitches for sarcosine, a prostate cancer biomarker. Two clones showed ß-galactosidase activities proportional to the concentration of sarcosine (A6 and C9) and are currently tested for in vitro binding assays.
|Chemistry and Biochemistry
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- Doctoral Dissertations